Method for measuring resistance of a patient HIV-2 to protease inhibitors

ABSTRACT

A search method in a biological sample containing an HIV 2 viral strain for possible resistance of said strain to treatment by an anti-protease agent, and nucleotide probes for the implementation thereof. According to methods known per se, the presence of at least one mutation at certain, specified, particular positions of the proteinic sequence of the protease of said viral strain from a biological sample taken from a patient contaminated by HIV 2 is searched. If said mutation is observed, the existence of a resistance to said anti-protease agent is assumed in the patient.

This is a Division of application Ser. No. 10/865,889 filed Jun. 14,2004, which in turn is a Division of application Ser. No. 09/980,777issued as U.S. Pat. No. 6,794,129 B1, which is a National Stage ofApplication No. PCT/FR00/01728 filed Jun. 21, 2000. The disclosure ofthe prior applications is hereby incorporated by reference herein in itsentirety.

BACKGROUND

The invention relates to a method for testing the resistance of theHIV-2 virus to antiprotease treatment in a patient infected with HIV-2as well as nucleotide probes usable for such testing.

Acquired immunodeficiency syndrome (AIDS) is caused by two viruses:HIV-1 and HIV-2. HIV-1 is present throughout the world while HIV-2 ispresent mainly in western Africa.

Effective antiviral treatments have been in widespread use since 1996 indeveloped countries where the virus present is HIV-1. Because of theircost, these treatments cannot be used in developing countries whereHIV-2 is present.

There are three types of antiretroviral treatments: antiprotease(Indinavir, Ritonavir, Saquinavir, Nelfinavir, and Amprenavir),nucleoside reverse transcriptase (RT) inhibitors (Zidovudine,Didanosine, Zalcitabine, Lamivudine, Stavudine, Abacavir, FTC, andAdefovir), and nonnucleoside RT inhibitors (Nevirapine, Delavirdine, andEfavirenz). These treatments are often given in combination; this isknown as multiple-drug therapy.

Antiproteases elicit primary mutations which confer a high degree ofresistance but alter the ability of the virus to replicate. Thus, thevirus needs to select secondary mutations if it is to be both resistantand able to replicate actively. Also, reverse transcriptase mutationshave been described where nucleoside RT inhibitors have been used incombination.

During treatment with HIV-1 infection, particularly if the levels ofdrug in the bloodstream are inadequate, viral replication isinsufficiently inhibited, or rises above the detection threshold ofavailable viral load techniques (the “viral load” measures the quantityof virus genomes in the bloodstream). Because of the high error rate ofreverse transcription, mutations take place in the genes targeted byprotease and reverse transcriptase treatment. Certain mutations bringabout various degrees of resistance to antivirals. Virologic failureoccurs in 20 to 40% of patients treated with current multiple drugregimens.

If viruses resistant to one or more substances can be shown for apatient before treatment or if the viral load increases again, the bestdrug combination for treating HIV-1 can be chosen.

There are currently no published data on mutations in the HIV-2 genomedue to the use of antiproteases.

Antiprotease agents that are active against HIV-1 are also activeagainst HIV-2. However, there are no methods available to assist theclinician in determining resistance to antiprotease drugs in patientsinfected with HIV-2.

SUMMARY

The amino acid sequence of the HIV-2 protease is known. In the presentapplication, the numbering system for this amino acid sequence can bededuced from that described in Human Retroviruses and Aids, 1997, LosAlamos National Laboratory, Los Alamos, N. Mex., Chapter II, pp. B10 andB11. The first amino acid in the protease sequence, considered to beposition 1 in the present application, is the proline in position 86 ofthe polyprotein PoL in the ROD strain.

It has now been discovered that antiprotease drugs can bring aboutmutations in positions 45, 54, 64, 84, and 90 and in positions 10, 46 ofthe HIV-2 protease and that the mutated viral strains thus appearing areusually resistant to at least one of the antiprotease drugs used.

Hence, the subject of the present invention is a method for testingresistance of an HIV-2 viral strain to antiprotease treatment.

In a preliminary testing phase, this is a method wherein:

a) using known methods, the presence of at least one mutation at one ofpositions 45, 54, 64, 84, and 90 or one of positions 10, 46 of theprotein sequence of the protease of said viral strain in a biologicalsample taken from a patient infected with HIV-2 is investigated,

b) of the mutations founds in a), those which, after cloning in an HIV-2virus, do not prevent the virus clone obtained from multiplying inculture in the presence of said antiprotease drug are selected, and

c) if at least one mutation is selected at step b), it is concluded thatresistance exists to the antiprotease drug referred to in b).

Of the mutations found in a), those which, when present in a gene clonedin an HIV-2 virus, cause the viral clone not to be significantlyprevented from multiplying in the presence of said antiprotease drug areselected. The following procedure may be used to implement step b). Themutations tested are inserted individually into a viral clone bymutagenesis directed by the method described in the article by Kemp etal., J. Virol., 72(6), pp. 5093-5098, 1998. The clones thus obtained arecultured with a “wild-type” (i.e. non-mutated) virus clone as areference in the presence of the various antiprotease drugs able to actagainst the HIV-2 virus. By measuring the IC₅₀ with a calorimetric testfor example (see above Kemp article), the size of the mutation (minor ormajor) with the various drugs tested can be determined. Thus, one canselect the mutations that allow the virus to multiply in the presence ofan antiprotease agent as these mutations give rise to strains resistantto this antiprotease.

Of course, in the case outlined in c), the future treatment planned forthe patient would be a different antiprotease agent from the agent shownto elicit resistance in this patient.

If step b) did not select a mutation found in step a), it may beconcluded that the mutation in question did not elicit resistance by theHIV-2 virus to the antiprotease drug tested for the patient in question.

Obviously, when step b) identifies a mutation that generates resistanceto a given antiprotease drug, step b) of the method described above neednot be carried out in the future. In this case, step a) would sufficebecause the link between the mutation and resistance to the antiproteasedrug would be established once and for all; one can go on directly tostep c) and conclude that there is resistance to the antiprotease agentstudied.

The invention relates in particular to a method for detecting anyresistance of an HIV-2 viral strain to treatment by an antiprotease drugin which the presence of at least one mutation chosen from the followingmutations:

-   -   K 45 R, I 54 M, I 64 V, I 84 L and L 90 M, or V 10 I, I 46 V,        and I 82 M,        in the protein sequence of the protease of said viral strain is        investigated and in which said resistance is concluded to exist        if said mutation or said mutations is or are present.

The conventional notation in the present application for describing amutation is as follows: The number indicates the position in the aminoacid sequence of the HIV-2 protease. The letter to the left of thenumber is the amino acid of the wild-type strain in the internationalclassification, with the one-letter code. The letter to the right of thenumber is the amino acid, in the same classification, resulting from amutation.

“Wild-type strain” is understood to be a viral strain that has notmutated after treatment with an antiprotease.

To identify a mutation in the protein sequence of the protease of theviral strain in question, it is preferable to look for a correspondingmutation in the nucleotide sequence of the gene of said protease. Thesemutations can be tested on the DNA or the RNA. Of course, in looking fora mutation in the protein sequence by seeking a mutation in thenucleotide sequence, degeneration of the genetic code would be takeninto account, namely a given amino acid can be coded by differentcodons. This mutation assay can be done in the nucleotide sequence byknown methods, particularly by hybridization or sequencing techniques.

DETAILED DESCRIPTION OF EMBODIMENT

In a first embodiment of the invention, a hybridization method usingspecific probes is implemented to test for the mutation or mutations.

A particular embodiment using a hybridization method consists ofobtaining a polynucleotide containing all or part of the protease gene,and including the sequence of interest corresponding to the regioncontaining the mutation to be assayed. Such a polynucleotide can beobtained in particular by enzymatic amplification. The method used inthis case comprises the steps consisting of placing said polynucleotidein contact with a nucleotide probe that is attached or attachable to asolid substrate and is able to hybridize specifically with such apolynucleotide only if the polynucleotide has the mutation studied; thenrevealing the presence of the polynucleotide attached to the solidsubstrate by the capture probe, using known methods. For this purpose,the solid substrate can be washed, after which the presence of thepolynucleotide, attached to the substrate, is revealed either by aphysical method or with an appropriate marker.

The probe can be attached directly by adsorption or by covalence. It canalso be attached indirectly by a ligand/antiligand-type reaction such asthe biotin/streptavidin or haptene/antibody pair, with the antiligandattaching to the solid substrate and the ligand to the probe, forexample.

The polynucleotide can also be labeled during the enzymaticamplification stage, for example using a triphosphate nucleoside labeledfor the amplification reaction. The labeled nucleotide will be adeoxyribonucleotide in amplification systems generating a DNA, such asPCR, or a ribonucleotide in amplification techniques generating an RNA,such as the TMA or NASBA techniques.

The polynucleotide can also be labeled after the amplification stage,for example by hybridizing a probe labeled by the sandwich hybridizingtechnique described in document WO 91/19812.

A particular method of labeling polynucleotides is described inapplication FR 98 07870 by the applicant.

Alternatively, the polynucleotide including all or part of the proteasegene can be prepared by enzymatic amplification, elongating primers thathave a ligand. The polynucleotide obtained, which will thus contain theligand, can be attached to the solid substrate by interaction with acorresponding antiligand. The solid substrate to which thepolynucleotide is attached is then placed in contact with at least oneprobe able to attach specifically to the polynucleotide only if itcontains the sought-after mutation. If this mutation is present, theprobe will be attached to the solid substrate by the hybrid it formswith the polynucleotide, which itself is attached. One need then onlyreveal the presence of the hybrid so formed by known methods.

In another embodiment, a hybridization method is used that comprises thesteps of enzymatically amplifying all or part of the protease gene usingprimers carrying a ligand to generate a polynucleotide having at leastone ligand, attaching the polynucleotide to a solid substrate byinteraction with an antiligand as described above, placing said attachedpolynucleotide in contact with at least one probe capable of hybridizingspecifically therewith, and revealing the hybrid formed, if any. Theprobe must hybridize only if the polynucleotide contains thesought-after mutation.

Other detection methods by hybridization may be considered such as thatdescribed in Kricka et al., Clinical Chemistry, 45(4), pp. 453-458, 1999or Keller G. H. et al., DNA Probes, 2nd Ed., Stockton Press, 1993,sections 5 and 6, pp. 173-249.

The “solid substrate” as used here includes all the materials on which apolynucleotide can be immobilized. Synthetic materials or naturalmaterials, that may be chemically modified, can be used for the solidsubstrate, particularly polysaccharides such as cellulose-basedmaterials, for example paper, cellulose derivatives such as celluloseacetate and nitrocellulose, or dextran; polymers, copolymers,particularly those based on styrene-type monomers, natural fibers suchas cotton, and synthetic fibers such as nylon; minerals such as silica,quartz, glasses, and ceramics; latexes; magnetic particles; metalderivatives; gels; etc. The solid substrate may be in the form of amicrotitration plate, a membrane as described in application WO 9412670,a particle, or a biochip.

“Biochip” is understood to be a solid substrate of small size to which aplurality of polynucleotides are attached at predetermined positions.

Examples of these biochips are given for example in the publications ofG. Ramsay, Nature Biotechnology, 16, pp. 40-44, 1998; F. Ginot, HumanMutation, 10, pp. 1-10, 1997; J. Cheng et al., Molecular Diagnosis,1(3), pp. 183-200, 1996; T. Livache et al., Nucleic Acids Research,22(15), pp. 2915-2921, 1994; J. Cheng et al., Nature Biotechnology, 16,pp. 541-546, 1998. The main property of the solid substrate must be topreserve the hybridization properties of the probes on the target andallow a minimum background noise for the detection method. One advantageof biochips is that they simplify the use of numerous probes, takinginto account the polymorphism of the virus in areas abutting thesought-after mutation. A biochip for verifying the presence or absenceof mutations can be made by the procedure described by Kozal M. et al.,Nature Medicine, 2, pp. 753-759, 1996, as a function of alignments ofsequences known for different HIV-2 strains.

A “marker” is understood to be a tracer able to generate a signal. Anonexhaustive list of these tracers includes enzymes producing adetectable signal, for example by colorimetry, fluorescence, orluminescence, such as horseradish peroxidase, alkaline phosphatase,beta-galactosidase, glucose-6-phosphate-dehydrogenase; chromophores suchas fluorescent, luminescent, or dye compounds; electron density groupsdetectable by electron microscopy, or by their electrical propertiessuch as conductivity, by amperometry or voltametry methods, or byimpedance measurement; groups detectable by optical methods such asdiffraction, surface plasmon resonance, or variation in contact angle,or by physical methods such as atomic force spectroscopy, tunnel effect,etc.; radioactive molecules such as ³²P, ³⁵S, or ¹²⁵I.

Signal amplification systems can be used as described in document WO/9508000 and in this case, the preliminary enzymatic amplification reactionmay be unnecessary.

The term “primer” designates an oligonucleotide sequence able tohybridize to a useful nucleic sequence and to serve as a starting pointfor an enzymatic elongation reaction to produce a nucleic acid fragmentcomplementing a target of interest such as the gene of the protease orpart of this gene. The primer has a size of between 5 and 50nucleotides, particularly between 10 and 30 nucleotides. Preferably, theprimers are chosen in the preserved regions of the HIV-2 virus to enableall the viral strains that could be encountered in a patient to beamplified in order to deal with the polymorphism inherent in the HIV-2virus.

The probes for demonstrating mutations at positions 45 and/or 54 and/or64 and/or 84 and/or 90, as well as those showing mutations at positions10 and/or 46 by hybridizing on all or part of the protease gene of theHIV-2 virus present in a biological sample, are also a subject of thepresent invention.

The term “probe” refers to an oligonucleotide sequence able to hybridizespecifically with a nucleic acid sequence of interest. Here, since thegoal of the present invention is to detect a point mutation on the geneof the HIV-2 protease, the probe must be able to distinguish a pointmutation under predetermined hybridization or washing conditions. Thesize of these probes is between 5 and 40 nucleotides, particularlybetween 9 and 25 nucleotides. Methods for determining these probes havebeen described for example in the patent application WO 97/27332. Theprobe is, for example, constructed such that the position of themutation to be detected is substantially in the center of the probe.

The oligonucleotides used as primers or probes can include natural ormodified nucleotides such as phosphorothioates, H-phosphonates,alkylphosphorothioates, or analogs of nucleotides containing bases suchas inosine or nebularin in the place of the purine or pyrimidine basespresent in the A, T, C, G, and U nucleotides.

These primers or probes can be composed totally or partially of alpha orbeta anomerism nucleosides or isomers in the D or L series, or PNA(Nielsen et al., Nucleic Acid Research, 21(2), pp. 197-200, 1993).

In another embodiment of the invention, the mutation or mutations is/aredetected by sequencing all or part of the protease gene.

The various sequencing methods are well known: In particular, thesequencing methods of Sanger, the sequencing methods using four wells toreact the sequences studied with sequencing primers labeled by fourdifferent fluorophores (Perkin-Elmer “ABI Prism Dye Primer” procedure),or the method described in U.S. Pat. No. 5,795,722 (Visible Genetics),or the method using labeled nucleotides (Perkin-Elmer “ABI Prism DyeTerminator” procedure) instead of labeled primers can be used. Thesequencing methods are described, for example, in Molecular Cloning, ALaboratory Manual, Sambrook, Fritsch, and Maniatis, Cold Spring HarborLaboratory Press, 1989, Chapter 13.

In a particular embodiment of the invention, the presence of only one ormore given mutations is tested. In another embodiment of the invention,both the mutated nucleotide sequence and the wild-type (non-mutated)nucleotide sequence are tested. If a hybridization method is used, atleast two types of probes are defined for each position able to mutate:a probe type specific to the mutated sequence and a probe type specificto the wild-type sequence. Using both these types of probes enables themethod to be controlled, since at least one of the two probe types hasto react. Another advantage is to reveal the presence of mutated strainsand wild-type strains in a given patient, where present.

Preferably, the target nucleic acid is subjected to a preliminaryenzymatic amplification reaction to increase the sensitivity of thetest, but it is possible to detect the target nucleic acid directly. Thearticles by Lewis (1992, Genetic Engineering News, 12, 1-9), andAbramson and Myers (1993, Curr. Opin. Biotechnol., 4, 41-47) giveexamples of target amplification. The enzymatic amplification techniqueis, for example, chosen from the NASBA (Nucleic Acid Sequence BasedAmplification), TMA (Transcription Mediated Amplification), RT-PCR(Reverse Transcriptase-Polymerase Chain Reaction), SDA (StrandDisplacement Amplification), or LCR (Ligase Chain Reaction) techniques.

Mutant viral strains are detected from a possibly pretreated biologicsample. “Pretreatment” means the various steps by which the sample istreated to make the target nucleic acid, namely the protease gene,accessible, for example lysis, fluidization, concentration, or capture(see for example U.S. Pat. Nos. 5,750,388 and 5,766,849) using methodsknown of themselves.

To extract the viral RNA, one may, for example, use the reagent sold bythe Boeringher Mannheim Company (High Pure Viral RNA reference 1858882)or the Quiagen kit (Viral RNA reference 29504). Other procedures aredescribed in Maniatis et al., Molecular Cloning: A Laboratory Manual,2nd Edition, Cold Spring Harbor Laboratory Press, 1989. The biologicsample can be any sample from the human body or possibly a sampleenriched by culturing, such as blood, sperm, skin tissue,bronchoalveolar lavage fluid, biopsy, urine, colonies, liquid culture,etc.

The following examples illustrate the invention.

EXAMPLES Example 1

A study was conducted on three patients infected with HIV-2. Patients 1and 2 had never received antiproteases. Patient 3 had already receivedRitonavir for 8 months, and this treatment had been withdrawn 5 monthsbefore the study started. The first sample studied was taken before thebeginning of treatment in patients 1 and 2, and 6 weeks after the startof Ritonavir treatment for patient 3. In patients 1 and 2, samples taken2 and 5 months respectively after the start of treatment were studied.For patient 3, two samples (8 months and 11 months) were analyzed.Treatment consisted of Ritonavir for patient 2 and Ritonavir/Saquinavirfor patients 1 and 3, at the recommended doses.

Methods:

The plasmas were obtained by centrifuging whole blood at 800 g for 10minutes and clarified by a second centrifugation at 3000 g for 15minutes.

500 microliters of pure plasma were added to 1.5 milliliters of freshlymphocyte culture stimulated by PHA (10⁶ cells/dish). Viral replicationin the supernatant was monitored twice a week by measuring the level ofHIV P-24 antigen (ELAVIA Ag I, Sanofi Diagnostics Pasteur). The positivesupernatants were stored at −80° C. After ultracentrifugation of 1milliliter of supernatant (23,500 g for 1 hour), the HIV-2 RNA wasextracted by means of the Amplicor HCV Specimen Preparation kit (Roche).

The protease gene was retrotranscribed from 10 microliters of viral RNAsolution and amplified with the Titan One Tube RT-PCR kit (RocheMolecular Diagnostics). Reverse transcription and the firstamplification were done with the 3′ Prot and 5′ RT 3 primers (seebelow). The reaction at 50° C. for 30 minutes was followed by adenaturing step at 94° C. for 5 minutes then 40 cycles (30 seconds at94° C., 30 seconds at 55° C., 90 seconds at 68° C.), and finally at 68°C. for 7 minutes. The step PCR stage was done with 5 microliters of theproduct of the first stage with primers 3′ RTD and 5′ Prot 2.1, with 5minutes denaturing at 94° C., followed by 30 cycles (30 seconds at 94°C., followed by 30 cycles at 55° C., and 30 seconds at 72° C.) andfinally 10 minutes at 72° C. The primer sequence is the following:

(SEQ ID NO: 1) 3′Prot: CAGGGGCTGACACCAACAGCACCCCC (SEQ ID NO: 2) 5′RT 3:CCATTTTTTCACAGATCTCTTTTAATGCCTC (SEQ ID NO: 3) 3′RTD:ATGTGGGGGTATTATAAGGATTT (SEQ ID NO: 4) 5′Prot 2.1: GAAAGAAGCCCCGCAACTTC

The amplification products were purified with the QUIACQUICK PCRpurification kit (Quiagen) and sequenced directly with the 3′ RTD and 5′Prot 2.1 primers using the ABI Prism Dye Terminator Cycle Sequencing kit(Applied Biosystem). They were analyzed with the Applied Biosystem 377automatic sequencer and the sequences were aligned with the HIV-2consensus sequences (subtypes A and B).

Results:

Before treatment, no mutation was detected relative to the HIV-2 A and Bconsensus sequences.

After treatment, the following mutations were observed:

-   -   position 45: In patients 1 and 2, coexistence of a non-mutated        population (lysine; codon AAA) and a mutated population        (arginine; codon AGA) were observed, namely the K 45 K/R        mutation was observed;    -   position 54: In patients 1 and 2, replacement of isoleucine        (codon ATA) by methionine (codon ATG), namely the I 54 M        mutation, was observed;    -   position 64: A non-mutated population was observed, and a        population in which isoleucine (codon ATA) was replaced by a        valine (codon GTA), namely mutation I 64 I/V;    -   position 84: In patient 3, a non-mutated population (isoleucine        (codon ATC) and a mutated population with replacement of        isoleucine by a leucine (codon CTC) were observed, namely the I        84 I/L mutation;    -   position 90: In all 3 patients, replacement of leucine (codon        CTG) by a methionine (codon ATG) was observed, namely the L 90 M        mutation.

Similarly, the following mutations were observed:

-   -   position 10: Replacement of valine (codon GTA) by an isoleucine        (codon ATA): i.e. mutation V 10 I when a patient was treated        with Ritonavir;    -   position 46: Replacement of isoleucine (codon ATA) by a valine        (codon GTA): i.e. mutation I 46 V when a patient was treated        with Ritonavir; and

position 82: Replacement of isoleucine (codon ATA) by a methionine(codon ATG): i.e. mutation I 82 M when a patient was treated withIndinavir.

Example 2 Example of Probes that can be Used for Detecting Mutations onthe HIV-2 Protease Gene

The probes usable for revealing any mutations according to theinvention, using hybridization techniques, can be defined fromalignments published by Myers G. et al., 1997, Human Retroviruses andAIDS: A compilation and analysis of nucleic acid and amino acidsequences, Los Alamos National Laboratory, Los Alamos, N. Mex.

Of course, in addition to the probes expressly defined below, theinvention also includes equivalent nucleotide probes, namely probes ableto detect the same mutations on the protein sequence of the protease asthose detected by the probes defined below, taking into accountdegeneration in the genetic code, namely the fact that a given aminoacid can be coded by different codons.

Thus, the expression “equivalent nucleotide sequences” means anynucleotide sequences that differ from each other by at least onenucleotide but whose translation leads to the same protein sequence, inother words all nucleotide sequences coding for the same proteinsequence.

Of course, this comment applies to each codon in each probe. Thus, forexample, the amino acid in position 53 of HIV-2 protease is aphenylalanine which can be coded either by codon TTT or by codon TTC.The probes corresponding to each of these possibilities are of coursepart of the invention.

Depending on the particular hybridization conditions, particularly thetemperature and composition of the hybridization and washing buffers, itis possible to define probes that must include at least one of theminimum sequences below, or their complementary sequences. Probescontaining these sequences enable mutations to be distinguished in ahybridization process. Of course, analogous probes, obtained inparticular by introducing base analogs such as inosine or nebularin inpositions where polymorphism due to intrinsic variability of the virusis present lead to a similar result and are also part of the invention.

These sequences are given in the 5′ to 3′ direction:

CCA AAA ATA for a wild-type form of position 45.

CCA AAA GTA for a wild-type form of position 45.

CCT AAA ATA for a wild-type form of position 45.

CCA AGA ATA for a mutated form of position 45.

CCA AGA GTA for a mutated form of position 45.

CCT AGA ATA for a mutated form of position 45.

TTT ATA AAC for a wild-type form of position 54.

TTT ATA AAT for a wild-type form of position 54.

TTT ATG AAC for a mutated form of position 54.

TTT ATG AAT for a mutated form of position 54.

GAA ATA AAA for a wild-type form of position 64.

GAA ATA GAA for a wild-type form of position 64.

GAA GTA AAA for a mutated form of position 64.

GAA GTA GAA for a mutated form of position 64.

AAC ATC TTT for a wild-type form of position 84.

AAC ATT TTT for a wild-type form of position 84.

AAC CTC TTT for a mutated form of position 84.

ATT CTG ACA for a wild-type form of position 90.

ATC CTG ACA for a wild-type form of position 90.

ATT CTA ACA for a wild-type form of position 90.

ATC CTA ACA for a wild-type form of position 90.

ATT ATG ACA for a mutated form of position 90.

ATC ATG ACA for a mutated form of position 90.

or:

CCA GTA GTC for a wild-type form of position 10.

CCA ATA GTC for a mutated form of position 10.

AAA ATA GTA for a wild-type form of position 46.

AAA GTA GTA for a mutated form of position 46.

CCA ATC AAC for a wild-type form of position 82.

CCA ATA AAC for a wild-type form of position 82.

CCA ATG AAC for a mutated form of position 82.

To obtain probes longer than those with the minimum sequences of 9nucleotides shown above, it is of course necessary to choose additionalnucleotides in order to respect the sequence of the adjacent regions oneither side of the minimum sequence in the gene of the protease of anHIV-2 strain. These sequences can be obtained from databases.

For example, the probes indicated below, or their complementary probes,can be used.

(a) Position 45:

A probe having for example 9 to 25 nucleotides (preferably distributedsymmetrically about the mutated codon AGA) whose sequence is included inone of the following sequences:

ATTACACTCCAAGAATAGTAGGGGG (SEQ ID NO: 5) ATTATAGCCCAAGAATAGTAGGGGG (SEQID NO: 6) ATTATAGTCCAAGAATAGTAGGGGG (SEQ ID NO: 7)ATTATACCCCAAGAATAGTAGGGGG (SEQ ID NO: 8) ATTATAGTCCAAGAATAGTAGGAGG (SEQID NO: 9) ATTATACCCCAAGAATAGTAGGAGG (SEQ ID NO: 10) can be used.

(b) Position 54:

A probe having for example 9 to 25 nucleotides (preferably distributedsymmetrically about the mutated codon ATG) whose sequence is included inone of the following sequences:

TAGGGGGATTTATGAACACCAAAGA (SEQ ID NO: 11) TAGGGGGATTCATGAACACCAAAGA (SEQID NO: 12) TAGGAGGATTCATGAACACCAAAGA (SEQ ID NO: 13)TAGGAGGGTTCATGAACACCAAAGA (SEQ ID NO: 14) can be used.

(c) Position 64:

A probe having for example 9 to 25 nucleotides (preferably distributedsymmetrically about the mutated codon GTA) whose sequence is included inone of the following sequences:

AAAATGTAGAAGTAAAAGTACTAAA (SEQ ID NO: 15) AAAATATAGAAGTAAAAGTACTAAA (SEQID NO: 16) AAGATGTAGAAGTAAAGGTACTAAA (SEQ ID NO: 17)AAAATGTAGAAGTAGAAGTTCTAAA (SEQ ID NO: 18) AAAATGTAGAAGTAGAAGTCCTGGA (SEQID NO: 19) AAAGTGTAGAAGTAAGAGTGCTAAA (SEQ ID NO: 20) can be used.

(d) Position 84:

A probe having for example 9 to 25 nucleotides (preferably distributedsymmetrically about the mutated codon CTC) whose sequence is included inthe following sequence:

CCCCAATCAACCTCTTTGGCAGAAA (SEQ ID NO: 21) can be used.

(e) Position 90:

A probe having for example 9 to 25 nucleotides (preferably distributedsymmetrically about the mutated codon ATG) whose sequence is included inone of the following sequences:

GCAGAAATATTATGACAGCCTTAGG (SEQ ID NO: 22) GCAGAAATATTATGGCAACCTTAGG (SEQID NO: 23) GCAGAAATGTTATGACAGCTTTAGG (SEQ ID NO: 24)GCAGAAATATCATGACAGCCTTGGG (SEQ ID NO: 25) GCAGAAACATTATGACAGCCTTA (SEQID NO: 26) can be used.

1. A method for testing, in a biological sample from a patient infectedby HIV-2 containing at least one HIV-2 viral strain, the resistance ofsaid HIV-2 viral strain to treatment with an antiprotease agent,comprising investigating the presence of a mutation at position 82 ofthe protein sequence of the protease of said viral strain, said mutationhaving previously been found to elicit said resistance, and if such amutation is found, concluding that a viral strain resistant to saidantiprotease agent is present in the patient in question.
 2. The methodaccording to claim 1, wherein: a) the presence of a mutation at position82 of the protein sequence of the protease of said viral strain in abiological sample taken from a patient infected with HIV-2 isinvestigated, b) a mutation found in a) which, after cloning in an HIV-2virus, does not prevent the virus clone obtained from multiplying inculture in the presence of said antiprotease drug is selected, and c) ifthe mutation is selected at step b), it is concluded that resistanceexists to the antiprotease drug referred to in b).
 3. The methodaccording to claim 1, wherein the presence of the mutation I82M, in theprotein sequence of the protease to said viral strain is investigatedand in which said resistance is concluded to exist if said mutation ispresent.
 4. The method according to claim 1, wherein, to detect amutation of the protein sequence of the protease, a correspondingmutation is sought in the nucleotide sequence of the gene of saidprotease.
 5. The method according to claim 4, wherein said test iscarried out using hybridization techniques.
 6. The method according toclaim 4, wherein said test is carried out using sequencing techniques.7. The method of claim 4, wherein said mutation in the sequence of thegene corresponds to I82M.
 8. The method of claim 7, wherein saidmutation corresponds to a codon for position 82, which is ATG, insteadof ATA or ATC.
 9. The method of claim 1, said method further comprisinginvestigating the presence of at least one additional mutation at one ormore of positions 10, 45, 46, 54, 64, 84 and 90 of the protein sequenceof the protease of said viral strain.
 10. The method of claim 9, whereinthe at least one additional mutation is selected from the followingmutations: V10I, K45R, I46V, 154M, I64V, I84L and L90M.
 11. The methodof claim 9, wherein, to detect the at least one additional mutation ofthe protein sequence of the protease, at least one correspondingmutation is sought in the nucleotide sequence of the gene of saidprotease.
 12. The method of claim 9, wherein said additional mutation isat said position
 10. 13. The method of claim 9, wherein said additionalmutation is at said position
 45. 14. The method of claim 9, wherein saidadditional mutation is at said position
 46. 15. The method of claim 9,wherein said additional mutation is at said position
 54. 16. The methodof claim 9, wherein said additional mutation is at said position
 64. 17.The method of claim 9, wherein said additional mutation is at saidposition
 84. 18. The method of claim 11, wherein said additionalmutation corresponds to a condon for position 45, which is AGA, insteadof AAA.
 19. The method of claim 11, wherein said additional mutationcorresponds to a condon for position 54, which is ATG, instead of ATA.20. The method of claim 11, wherein said additional mutation correspondsto a condon for position 64, which is GTA, instead of ATA.
 21. Anucleotide probe usable in the method according to claim 1, comprising,as a minimum sequence, a sequence selected from the group consisting of:a) CCA ATG AAC possibly supplemented by the nucleotide sequence of anadjacent region of the gene of said protease, on either side of theminimum sequence, b) a nucleotide sequence equivalent to a sequencedefined in (a), and c) a sequence complementary to a sequence defined in(a) or in (b).